Bioelectric patterning during oogenesis: stage-specific distribution of membrane potentials, intracellular pH and ion-transport mechanisms in Drosophila ovarian follicles

Many aspects of patterning, axis formation and morphogenesis have been intensely studied in Drosophila ovarian follicles. Oogenesis involves hierarchically organized symmetry-breaking events [53], diverse reorganizations of the cytoskeleton [54,55], directed localization of cytoplasmic determinants [56], determination of cell fates and coordinated cell migrations [14-16]. All of these processes are mediated by successive interactions of signaling mechanisms, including cell contacts by cadherines and gap junctions, secreted signals, like EGF- and BMP-related proteins, and Notch signaling [57].

During S9 to S10B, we found striking stage-specific Vmem- and pHi-patterns that correspond to populations of morphologically distinguishable FC types. In addition, a prominent anteroposterior pHi-gradient was observed within the NC cluster. We also revealed, along the longitudinal and/or transversal follicle axes, asymmetric distributions of membrane channels, namely V-ATPases, L-type Ca2+-channels, amiloride-sensitive Na+-channels and NHE, and innexin-3-containing gap junctions, that all are supposed to be involved in the regulation of Vmem and/or pHi. Actually, we found remarkable accordance between the stage-specific distributions of these membrane channels and the Vmem- and pHi-patterns.

S9 and S10A are characterized through longitudinal bioelectric patterns, where the mFC are hyperpolarized in relation to the tFC, cFC and sFC. While the PC and the migrating BC are more depolarized, the BC are more acidic, and the tFC are more alkaline than the other FC. In addition, we found bioelectric gradients in the Ooc, with the posterior region being depolarized and alkaline in relation to the anterior region. S10B, however, is characterized through a transversal Vmem-pattern, with the ventral side of the FC epithelium being more depolarized than the dorsal side.

Close accordance was observed between the patterns of Vmem and the activity patterns of voltage-dependent L-type Ca2+-channels. All regions being more depolarized exhibited intense verapamil-FL fluorescence, indicating high concentrations of channels in the activated, or open (and inactivated) state, which depends on a depolarized Vmem. This finding points to a mechanism by which alterations of Vmem could be transduced into cellular responses through voltage-dependent Ca2+-currents. Equally distributed Ca2+-channels (indicated by Anti-Cavα1) are supposed to respond to stage-specific regional changes of Vmem and, thereby, regulate intracellular Ca2+-concentrations in discrete follicle regions. Ca2+-ions are known to act as signaling molecules in many developing systems e.g. [58,59]. Ca2+-signaling in the FC epithelium of Drosophila has not been addressed so far, but influences on the cytoskeleton, e.g. on contractions of the basal actomyosin network [60], seem to be likely.

Using antisera raised against different components of V-ATPases, we revealed an asymmetric distribution of proton pumps in the FC epithelium. During S9 to S10B, V-ATPases are accumulated in the lateral and apical plasma membranes on the ventral side of the follicle, including tFC, mFC, cFC and sFC. Since the activity of V-ATPases in epithelia is electrogenic, the pumps are supposed to have direct or indirect influence on Vmem in ovarian follicles. This hypothesis is supported by the accordance between the dorsoventral V-ATPase-pattern during S9 to S10B and the dorsoventral Vmem-pattern in S10B. Since, besides their primary functions, V-ATPases are also known to play specialized roles in developing systems e.g. [29], their accumulation on one follicle side points to a possible role during the regulation of the spatial coordinates.

Amiloride-sensitive Na+-channels and NHE showed high concentrations in anterior and low concentrations in posterior NC. This gradient corresponds to a pHi-gradient with a most alkaline anterior NC and a most acidic posterior NC adjacent to the anterodorsal region of the Ooc. Therefore, an interrelation between amiloride-sensitive Na+-transport and pHi is very likely. Either Na+-transport, probably through NHE in exchange to H+, is involved in the establishment of the pHi-gradient, or the pHi-gradient exerts influence on the activity of Na+-transporters. Despite cytoplasmic coupling via ring canals and gap junctions, each NC regulates its pHi independently. The resulting anteroposterior pHi-gradient is supposed to play a role in various processes during oogenesis. In general, pHi is known to exert influence on cell metabolism, enzyme activity, contractibility of cytoskeletal structures, cell polarity and proliferation rates of cells [61-63]. During S9, the pHi-gradient might also provide guidance cues for BC migration.

Of the ovarian gap-junction proteins innexin 1 to 4, only innexin 3 showed a striking non-uniform distribution within the FC epithelium: it is enriched in the lateral membranes of cFCs. This finding indicates distinct coupling conditions between these cells, concerning e.g. regulatory signals, Vmem and pHi. Moreover, innexin 3 seems to be involved in maintaining tissue integrity in response to tension [64], a function that is particularly important for cFC.

The cFC are specialized in several further respects: they are longer dye-coupled to the Ooc by gap junctions [12], they contain higher amounts of Na+,K+-ATPase [26,65] (which becomes activated under alkaline conditions [66]), and they possess a higher [Ca2+]i than the remaining FC [67]. The permeability of innexin channels in general has been reported to be Vmem-, pHi-, K+- and Ca2+-sensitive [68,69]. Restriction to distinct cell populations and independent regulation of permeability of specialized gap-junction channels (formed by different proteins in either homomeric, heteromeric or heterotypic combinations [50]) could provide mechanisms to generate or maintain boundaries between different FC populations.

Spatial patterns of Vmem and pHi related to non-uniform distribution and activity patterns of membrane channels (for summary, see Figure 8) are supposed to generate bioelectric signals during several important steps of oogenesis, e.g. for the regulation of spatial coordinates, migration processes, or reorganization of the cytoskeleton. In recent years, the complex interrelations as well as the physiological and cellular functions of bioelectric phenomena have become increasingly challenging [70]. Analysing the distribution and activity patterns of further ion-transport mechanisms - also in relevant Drosophila mutants e.g. [53,71,72] - as well as specific manipulations of Vmem and pHi by using appropriate inhibitors will be promising ways to help cracking the bioelectric code [73].

We are indebted to Reinhard Bauer and Michael Hoch (Bonn, Germany) for providing the innexin antisera, to Bernd Walz and Otto Baumann (Potsdam, Germany) for providing the V-ATPase antiserum, and to Maria Bugaro, Andrzej Steckiewicz and Isabel Weiß for technical assistance. Financial support by RWTH Aachen University is acknowledged.